PCR-Based Identification of Integrons Types and Extended-Spectrum Β-Lactamase Genes in Salmonella Species Collected from Pediatric Diarrheal Samples

Objective: Salmonella is a significant pathogen between food-borne diseases. Salmonella spp. strains that produce extended-spectrum β-Lactamases (ESBL) have become a medical problem for both antimicrobial therapy and infection control programs. The aim of this project was detection of ESBL genes and class I, II and III Integrons in the Salmonella isolates. Methods: 405 non-duplicative stool samples were obtained. Antibacterial susceptibility was defined by the disk diffusion and also double disk synergy test (DDST) was used for confirming of ESBL phenotype. The multiplex-PCRs were directed for recognition of ESBLs ( TEM , CTX-M and SHV ) and int (I, II, III) genes. Results: Out of 405 samples, 54 (13.4%) Salmonella were obtained. The highest resistance rate was related to the NA (51.8%), followed by SXT (50%), CTX (46.3%), and AMP (33.3%). DDST was conducted for all isolates and 7 (12.9%) Salmonella spp were ESBL positive. Molecular analysis showed that 5 (9.3 %) of isolates were carried blaTEM-1 which belonged to the S. infantis and S. typhimurium . Three (5.5%) non-typeable isolates and 2 S. typhimurium were positive for the CTX-M gene. The prevalence of different classes of integrons showed that 23 (42.5%) isolates carried the integrase ( int ) gene. Conclusion: This research demonstrates the predominant existence in the Salmonella of the TEM and CTX-M genes. So, class I integron were more prevalent than class II and III in Salmonella isolates. They are capable of transferring to bacteria of this genus and also the other genus of intestinal ones.


INTRODUCTION
Salmonella spp. are recognized as major food-borne pathogen in worldwide and its outbreaks are frequently related to the ingesting of contaminated food (1,2). In both developed and developing countries, human salmonellosis has been a major concern for public health. An estimated 800,000 to 4 million nontyphoidal salmonellosis (NTS) are recorded per year in the United States, and about 500 of them die (3). The most consequence of this infection is acute gastroenteritis and does not require to antibiotic therapy (4). However, chemotherapy is commonly suggested for cases with salmonellosis, mostly those cases at high risk of systemic infection, such as; elderly, pregnant, immunocompromised patients and children less than 1 year of age (3)(4)(5). The extensive misuse and misappropriation of antimicrobials in domestic animals and food additives have contributed to the increase of drug resistant pathogens such as Salmonella (6). Proper antibiotic therapy for non-typhoidal salmonellosis illnesses may reduce of severity and infection period and may also prevention of fatality and further illness transmission (7). The actual concerns are those strains that have acquired multiple drug resistance (MDR) against three or more classes of therapeutic agents (8). Extended-spectrum cephalosporins (ESCs) are counted as an alternative therapeutic choice for NTS infections that are resistant to treatment. The increased trend of using -lactam drugs to treat intestinal diseases had developed penicillin and cephalosporin resistance in Salmonella spp. in different parts of the world and had been a reason for drug therapeutic failure (DTF) (9,10). Usually, extended-spectrum beta-lactamases (ESBLs) are encoded by a large-sized plasmid moving from inter and intra species of bacteria (11). Resistance to ESCs is primiraliy promoted by generation of class A ESBLs, that capable of hydrolyzing oxyimino cephalosporins but are not active against carbapenems and cephamycins. Temoneira (TEM), cefotaximase (CTX-M) and sulfhydryl variable (SHV) are found in class A ESBLS. SHV type β-lactamases are associated with high ceftazidime resistance, but not with cefazolin and cefotaxime, whereas CTX-M β-lactamases are more efficient against cefotaxime.TEM β-lactamases,by comparison, impart resistance to oxyimino-βlactams groups, like ceftazidime, cefotaxime, and aztreonam (12,13). Instead of ESBLs, the mobile genetic elements (MGEs), including integrons, can evolve and distribute resistance genes in NTSs. Integrons are conserved segments (3ʹ-CS and 5ʹ-CS) genetic units categorized by their capacity to harbor and integrate gene cassettes by site-specific recombination. Three classes of antibiotic resistance integrons (ARIs) (classes I, II, and III) have been mostly recognized in the MDR phenotypes criteria and are identified according to integrase (int) genes (14, 15). The transferable class I (Tn21 and Tn402 derivatives) integrons are the most common type and followed by class II and III, respectively (16). Class I integrons harbor many antimicrobial gene cassettes encoding ESBLs, dfr, qacEΔ1, sul1 and aminoglycoside-modifying enzymes (AMEs) (17,18). Class II Integron located on the Tn7 and 3ʹ-CS contains five tns genes which are corresponding for the elasticity of transposons (TE). Integron of class III situated in a TE, but the 3ʹ-CS is unknown (19). The objective of the this research project was detection of blaTEM, blaCTX-M, blaSHV, and int genes (class I, II and III integrons) in the NTS strains isolated from in pediatric patients by multiplex PCR (M-PCR) and their antibiotic resistance profile.

Study design and Ethical approval
This work was financially supported by a grant (no. 724133739) by Babol University of Medical Sciences, Babol, Iran. Written informed consent form was collected from the patient's parents. Identifying information of each sample was kept secret.

Bacterial isolates
In the cross-sectional study, 405 non-duplicative stool samples were collected from the pediatric patients with aged less than 10 years. Samples were cultured on the Mac Conkey agar (Merck, Germany) and incubated at 37 °C for 24 h. Then, all suspected grown colonies were recognized as Salmonella by conventional biochemical and microbiological tests and confirmed by the API 20E system (Analytab, New York). Serotyping with specific O and H Salmonella antisera was performed according to the by slide agglutination method (Denka Seiken, Japan).

Antibiotic susceptibility test (AST)
Antibiotic susceptibility test was performed by disc diffusion (DD) method on the Mueller-Hinton Agar (Merck, Germany) plates according to the Clinical and Laboratory Standards Institute (CLSI M100-S28) guideline for the following antibiotics: amoxicillin/clavulanate (AMC; 20/10 μg), ciprofloxacin (CIP: 5 μg), amikacin (AK: 30  The combination disk test based on the clavulanic acid inhibitory effect was also used according to the CLSI guideline (13). E. coli ATCC 25922 was used as a reference strain for antimicrobial susceptibility test.

Molecular Analyses
M-PCRs were performed by using the DNA amplification instrument master cycler gradient(Eppendorf Co., Germany) for identification of ESBLs genes (blaTEM, blaCTX-M and blaSHV) and int genes (I, II and III). We used ESBLs and integrons primer as mentioned previously (12,20). DNA extraction for all Salmonella strains performed by using the boiling lysis method. Concisely, a loopful of bacterial colonies was suspended in the 1000 µl distilled water (DW) and boiled for 15 minutes and centrifuged at 7000×g for 5 minutes at 4°C and then cooling in ice for 15 minutes and centrifugation for 4 min at 8000×g. The DNA quality was measured by using the Nanodrop spectrophotometer (ND-1000; Thermo Scientific; Wilmington, DE, USA). M-PCR was done for amplification of ESBLs genes (blaTEM, blaCTX-M and blaSHV) in a volume of 1.3 µl of extracted genomic DNA was added to a final volume of 25 µl PCR reaction mixture comprising 2.0 µl of 10× PCR buffer, 1.5 µl MgCl2 (50 mM), 0.5 µl dNTPs (10 mM), 1.0 µl of each primer, 0.5 µl of Taq DNA polymerase (5 U/µl) (Amplicon Co., Denmark) and 13.2µl DW. The reaction mixture was done with the following procedure: denaturation at 94°C for 60 seconds, 33 cycles with denaturation at 94°C for 45 seconds, annealing at 59°C for 30 seconds, extension at 72°C for 60 seconds and final extension at 72°C for 5 minutes. So, amplification of int genes (I, II and III), the reaction mixture was completed in a thermal gradient cycler (Eppendorf Co., Germany) with the following PCR protocol: The cycling conditions were one cycle of 5 minutes at 95 °C; 30 cycles of 1 minutes at 95°C, 1 minutes at 65°C, and 1 minutes at 72°C; and one cycle of 10 minutes at 72°C. Briefly, the whole 25 μl volume of MPCR reaction mixture for all genes (intI, intII and intIII) contained of 1× PCR buffer, 3.0 mM of MgCl2, 2.0 mM of each dNTPs, 3U of Taq polymerase, 10 pmol of each primer, 2.5 μl of DNA template and distilled water to reach the reaction volume (12,20). The amplification products were separated by agarose gel electrophoresis by using 1.5% agarose gel stained with ethidium bromide (EtBr) and imagined with UV Gel documentation system (UVP Gel Seq Software, England).

Statistical analysis
The collected data were statistically analyzed using SPSS program (Version 16.0). Data were subjected to descriptive statistics and expressed in percentages.

DISCUSSION
In the present study, we performed antibiotic susceptibility and detection ESBLs and integron genes among Salmonella spp., isolated from pediatric feces samples. In total, 54 Salmonella spp., strains were analyzed throughout the project. Our prevalence of Salmonella spp., in stool samples was obtained 13.3%. These results are in agreement with the previous study (21). The present study demonstrated that typhoidal salmonellosis is more predominant than nontyphoidal salmonellosis and this result is like the reports by Sood et al. (22) on "Salmonellosis in developing countries". However, in Nigeria, NTS is more common than typhoidal salmonellosis. (21) Our results indicated the high frequency of S. Enteritidis strains in pediatric stool samples (n; 24, 44.5%), these findings was agreement with previous reports (21,(23)(24)(25). The higher number of Salmonella documented versus age-group 0 to 5 years and 6 to 15 years showed that salmonellosis manifested more in children than adults (21,(23)(24)(25). All Salmonella spp. collected was tested for antimicrobial susceptibility with 16 antibiotics in the present study. Of the total isolates, 51.8% were found to be resistant to NA, 50% resistant to SXT, 46.3% resistant to CTX, 33 (27,28). Ranjbar et al (20) reported that most prevalent resistances were associated with doxycycline (64.7%), NA (61.2%), TET (51.8%), and streptomycin (42.8%). Additionally, our 54 strains of Salmonella spp exhibited susceptible to the all antimicrobial agents used, such as CIP, OFX, IPM, TN and AK. In similarity to our study, 100 % susceptibility of Salmonella spp to the CRO, IPM and OFX were observed in several studies (20,(29)(30)(31) . In our study, 29 strains out of 54 (53.7 %) Salmonella spp showed either a class 1 integron or class 1 and 2 integron. While, in all strains tested by M-PCR ,integron class 3 was not detected. This distribution of our integron positive isolates approximately is consistent with previous reports that Salmonella carried high prevalence of integron class 1, lower class 2 and no class 3 (20,(32)(33)(34)(35). This incidence was higher in comparison with a study conducted by Cabrera et al. (36), who exhibited that 25% of S. Enterica confined class 1 integrons in Spain. Other research in the United Kingdom discovered that the occurrence of class 1 integrons was 20.4% in Salmonella spp. (14), 13% in Hong Kong (37), and 13% in Vietnam (38). In addition to integron genes, ESBLs gene was identified in 10 (18.5%) our isolates of Salmonella spp. and also the main enzymes found in them were CTX-M and TEM. While SHV was not developed by any clinical isolates. This prevalence is much higher than any frequency rates of Salmonella spp. reported, as recorded in Taiwan by 1.5 percent (39, 40), 1.9% in the USA (41), 2.4% in Latin America (42), 0.8% in Europe and 3.4% in the Western Pacific region (42). The reason behind this high incidence of ESBL-producing Salmonella in our study could be attributed to the different rates of antibiotic using in society (43). In 2005, Moubareck and colleagues (44) described for the first time an ESBL-producing S. Typhimurium of CTX-M-15-type in the Lebanon. None of our isolates included blaSHV, these findings are agreeing with the previous study (43). In Elumalai et al (12) study, blaTEM-1 was identified in 4.47% (n; 6/134) of Salmonella spp, which belonged to the S. Typhi serotype. All six TEM-positive strains were negative for the blaSHV gene and none of the isolates was found positive about the presence of the blaCTX-M gene.

CONCLUSION
As in our Salmonella strains, the high frequency of integron-positive isolates has shown that these mobile genetic factors are widespread among various Salmonella spp. and associated with reducing susceptibility for first-choice antibiotic therapy.

Conflict of interest
No conflict of interest was declared by the authors.